CN105325298B - A kind of lotus rhizome stem-tip tissue Initial culture method - Google Patents
A kind of lotus rhizome stem-tip tissue Initial culture method Download PDFInfo
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- CN105325298B CN105325298B CN201510800063.XA CN201510800063A CN105325298B CN 105325298 B CN105325298 B CN 105325298B CN 201510800063 A CN201510800063 A CN 201510800063A CN 105325298 B CN105325298 B CN 105325298B
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Abstract
The invention discloses a kind of lotus rhizome stem-tip tissue Initial culture method.Comprise the following steps：1) collection of lotus rhizome sprout；2) pre-treatment of sprout；3) sterilization and inoculation；4) stem-tip tissue expands culture；5) lamina culture.The present invention is ground by using suitable culture medium and the method for culture to prevent explantation tissue's brown stain；The generation of leaf malformation variation is prevented by using lamina culture medium, makes its cauline leaf normal development.This method can make the planting percent of lotus rhizome stem apex apical meristem reach more than 90%.
Description
Technical field
The present invention relates to a kind of lotus rhizome stem-tip tissue Initial culture method, belongs to agricultural, biological technical field.
Background technology
Plant Tissue Breeding refers to isolate the tissue to suit the requirements, organ or cell, protoplast etc. from plant, led to
Cross sterile working, cultivated under manual control condition with obtain regeneration intact plant or production with economic value its
The technology of his product.The technology is in quickly plant, plant virus-free and the plant species of breeding rare plant or high economic value
Preservation of matter resource etc. has wide practical use.
Lotus rhizome is asexually propagated plant, and maternal plant is also easy to produce subterranean stem, and new plant can be produced on rhizomatous section, numerous
Grow offspring.This propagation method, the disease of parent and physiological barrier etc. are easily passed into offspring, and can be accumulated by generation,
The yield and quality of Progeny plants is caused to decline.Artificial culture and propagation offspring is carried out using lotus rhizome stem-tip tissue, can effectively be prevented
The only propagation of pathogeny, it is ensured that offspring can keep good strains of seeds.Lotus rhizome stem tip tissue culture divides Initial culture, Multiplying culture, takes root
4 steps, wherein the stem-tip tissue Initial culture such as culture and domestication refer to lotus rhizome stem-tip tissue being inoculated into specific culture medium
Upper process of the acquisition without pathogeny plant, it is to obtain the important step without pathogeny plant, and the key of lotus rhizome stem tip tissue culture
Technology.
Problem present in lotus rhizome stem apex Initial culture is at present：Browning rate height easily occurs for stem-tip tissue after inoculation, newborn
The blade of leaf can not deploy, and petiole can not extend, and growth deformity, inoculation survival rate is low, and aberration rate is high, and planting percent is low.
The content of the invention
It is an object of the invention to provide a kind of lotus rhizome stem-tip tissue Initial culture method.This method can make lotus rhizome stem apex top
The planting percent of separate living tissue reaches more than 90%.
Applicant it has been investigated that：Lotus rhizome stem-tip tissue Initial culture process can be divided into explant and expand and cauline leaf growth
Two stages.
Existing lotus rhizome stem tip tissue culture method, using solid medium culture, explant easily occurs after inoculation
Necrosis.Study and test through applicant, find brown stain to explant of different types of culture medium and training method and swollen
Notable difference be present greatly, have developed lotus rhizome explant and expand culture medium, using the culture medium to carry out liquid concussion and cultivate can be with
The generation of brown stain is prevented, the browning rate of explant is reduced within 5%.
Existing lotus rhizome stem tip tissue culture method, dysplasia after stem-tip tissue inoculation, the blade of Newborn Leaves can not be opened up
Open, petiole can not extend, growth deformity.Applicant has found that culture medium deploys to lotus rhizome cauline leaf by substantial amounts of research and experiment
There is significant impact, have developed lotus rhizome stem-tip tissue lamina culture medium, 30- is cultivated in culture medium is expanded after explant inoculation
After 40d, it is transformed into lotus rhizome lamina culture medium and cultivates, lopsided generation can be prevented, the explantation tissue for enabling to expand normally gives birth to
Long development.
Based on the studies above, the present invention prevents that explantation tissue is brown by using suitable culture medium and the method for culture
Become；The generation of leaf malformation variation is prevented by using lamina culture medium, develops its cauline leaf；Its concrete technical scheme is as follows：
A kind of lotus rhizome stem-tip tissue Initial culture method, comprises the following steps：
1. the collection of lotus rhizome sprout：Acquisition time is the 5-8 months, chooses rhizomatous terminal bud；
2. the pre-treatment of sprout：Terminal bud is left and taken into top 3-5cm, is handled with 84 thimerosals and flowing water cleans；
3. sterilization and inoculation：Sterilization and inoculation are carried out in superclean bench, specific as follows：
3.1 sterilization：
3.1.1 with 75% alcohol disinfecting 30 seconds；
3.1.2 effective chlorine density is 1% liquor natrii hypochloritis, is sterilized 30 minutes；
3.1.3 it is standby after aqua sterilisa cleaning；
3.2 inoculation：
3.2.1 explant is won：In the case where multiplication factor is 10-40x anatomical lens, ecto-entad is divested outside growing point successively
The blade of parcel is enclosed, until growing point exposes.
3.2.2 organized with dissecting needle needle point picking growing point top, be inoculated into lotus rhizome and expand in culture medium：The group of culture medium
Cheng Shi：1/2MS+NAA (0.1-0.3mg/L)+BA (0.2-0.5mg/L)+sucrose (3%), pH 5.8-6.0；Culture vessel is
A diameter of 25mm test tube, culture medium dosage are 15-20ml.
4. stem-tip tissue expands culture：The test tube being inoculated with is placed on rotation concussion shaking table and carries out concussion and cultivate, vibration velocity
For 2rmp, 23-25 DEG C of condition of culture temperature, light intensity 2000-3000lx, light application time 16hr；Cultivation cycle 30-40d is formed
The sprout expanded；
5. lamina culture：
Stem-tip tissue is replaced with lamina culture medium, the group of culture medium after medium culture 30-40d is expanded, by culture medium
Into being MS+BA (0.1-0.3mg/L)+GA (0.2-0.5mg/L)+sucrose (5%), pH 5.8-6.0；Culture vessel is 300ml
Triangular flask, culture medium dosage 100ml, 23-25 DEG C of condition of culture temperature, light intensity 2000-3000lx, light application time 16hr, training
Support 40-50 days cycle.
In step 1), in order to ensure the purity of kind, sprout collection will be carried out in kind garden, acquisition length 5-
10cm, to ensure the quality of stolon.
Pre-treatment in step 2) is with 400 times of 84 medicining liquid dipping sprout 10-15 minute, then with distilled water flushing 10-
15 minutes, foregoing 400 times referred to water by 400：1 volume ratio dilutes 84 thimerosals.
It is easy to operate when being easy for sterilization inoculation from the reason for head clip stolon 3-5cm in step 2)；
Cleaned 3-5 times with aqua sterilisa in step 3).
The multiplication factor of anatomical lens described in step 3) is 10-40x, in order to clearly observe growing point, convenient sampling.
In step 3), to ensure explant size ﹤ 0.5mm, the dissecting needle pin rugosity is Φ=0.2-0.4mm, operation
When as contrast, needle point take growing point tissue be no more than pin rugosity.
In step 3), culture vessel is test tube Φ=2.5cm during the Initial culture, and often pipe culture medium addition is 15-
20ml。
The invention has the advantages that：
1st, expanding culture medium using liquid can accelerate to expand speed, and the generation of brown stain can be prevented by carrying out concussion and cultivate,
Ensure that explant survives and reach more than 90%.
2nd, the lamina culture medium used can enable the normal lamina of sprout that explantation tissue is formed, and prevent the hair of variation
It is raw, it ensure that the purity of offspring's kind property.
3rd, using rugosity Φ=0.2-0.4mm dissecting needle, as control during sampling, explant can effectively be controlled
Diameter is less than 0.5mm, and ensure that explant is without pathogeny, and its offspring bred is entirely without poisoning.
Brief description of the drawings
Fig. 1 is lotus rhizome stem apex pictorial diagram of the present invention.
Fig. 2 is that stem-tip tissue of the present invention expands culture pictorial diagram.
Fig. 3 is sprout lamina culture pictorial diagram of the present invention.
Fig. 4 is lotus rhizome Initial culture plant pictorial diagram of the present invention.
Embodiment
Embodiment
1st, the collection of sprout：Respectively choose lotus root varieties be ' E Lian 4 ', ' beauty is red ', ' floatd flower, and ' three kind are cultivated.
In order to ensure that the collection of the purity sprout of kind is carried out in kind garden, acquisition time is the 5-8 months, chooses the rhizomatous top of hypertrophy
Bud, field sampling sprout length is 5-8cm.
2nd, sprout is handled：The sprout of collection is used after originally rinsing 15-20 minutes, with 400 times of 84 medicining liquid dipping 10-15
Minute, then with distilled water flushing 10-15 minutes.
3rd, sterilization and inoculation：Sterilization and inoculation are carried out in superclean bench
3.1 sterilization：
3.1.1 by the sprout of above-mentioned processing, the clip 3-5cm since top.
3.1.2 with 75% the alcohol disinfecting 15-20 seconds；
3.1.3 effective chlorine density is 1% liquor natrii hypochloritis, is sterilized 30 minutes
3.1.4 aqua sterilisa is standby after cleaning 3-5 times.
3.2 inoculation：
3.2.1 explant is won：Ecto-entad divests the leaf of growing point periphery parcel successively under 10-40x anatomical lens
Piece, until growing point exposes.
3.2.2 organized with the pin rugosity Φ dissecting needle needle point picking growing point tops for being 0.2-0.4mm, be inoculated into culture medium
In, using dissecting needle as contrast during operation, needle point takes the growing point tissue to be no more than the rugosity of pin, the composition of culture medium：1/2MS
+ NAA (0.1-0.3mg/L)+BA (0.2-0.5mg/L)+sucrose (3%), pH 5.8-6.0；Culture vessel is a diameter of 25mm
Test tube, culture medium dosage is 15-20ml.
4th, stem-tip tissue expands culture：The test tube being inoculated with exists, and is placed on rotation concussion shaking table and carries out concussion and cultivate, shakes
Speed is 2rmp, 23-25 DEG C of condition of culture temperature, light intensity 2000-3000lx, light application time 16hr；Cultivation cycle 30-40d shapes
Into the sprout expanded；
5th, lamina culture：
Stem-tip tissue is replaced with lamina culture medium, the group of culture medium after medium culture 30-40d is expanded, by culture medium
Into being MS+BA (0.1-0.3mg/L)+GA (0.2-0.5mg/L)+sucrose (5%), pH 5.8-6.0；Culture vessel is 300ml
Blake bottle, culture medium dosage 100ml, 23-25 DEG C of condition of culture temperature, light intensity 2000-3000lx, light application time 16hr, training
Support 40-50 days and be achieved with primary plant.By above-mentioned incubation ' E Lian 4 ', ' beauty is red ' and ' float flower ' Initial culture
Seedling rate be respectively 93.5%, 92.6% and 97.8%.
Claims (8)
- A kind of 1. lotus rhizome stem-tip tissue Initial culture method, it is characterised in that comprise the following steps：1) collection of lotus rhizome sprout：Acquisition time is the 5-8 months, chooses rhizomatous terminal bud；2) pre-treatment of sprout：Terminal bud is left and taken into top 3-5cm, is handled with thimerosal and flowing water cleans；3) sterilization and inoculation：The inoculation method is：A. explant is won：Ecto-entad divests the blade of growing point periphery parcel successively under anatomical lens, until growing point Exposure；B. organized with dissecting needle needle point picking growing point top, be inoculated into lotus rhizome and expand in culture medium；The composition of the culture medium It is：1/2MS+NAA0.1-0.3mg/L+BA0.2-0.5mg/L+ sucrose 3%, pH 5.8-6.0；To ensure explant size ﹤ 0.5mm, the dissecting needle pin rugosity are Φ=0.2-0.4mm, and as contrast during operation, needle point takes growing point tissue not surpass Cross the rugosity of pin；4) stem-tip tissue expands culture：Concussion and cultivate, vibration velocity 2rmp, 23-25 DEG C of condition of culture temperature, light intensity are carried out after inoculation 2000-3000lx, light application time 16hr；Culture 30-40d forms the sprout expanded；5) lamina culture：Stem-tip tissue is replaced with lamina culture medium after medium culture 30-40d is expanded, by culture medium, culture The composition of base is MS+BA0.1-0.3mg/L+GA0.2-0.5mg/L+ sucrose 5%, pH 5.8-6.0；Condition of culture temperature 23- 25 DEG C, light intensity 2000-3000lx, light application time 16hr, cultivate 40-50 days.
- 2. according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterised in that step 1）In, in order to Ensure the purity of kind, sprout collection will be carried out in kind garden, acquisition length 5-10cm.
- 3. according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterised in that step 2）In sterilization Liquid processing and flowing water cleaning are with 400 times of 84 medicining liquid dipping sprout 10-15 minute, then with distilled water flushing 10-15 minutes, institute 400 times are stated to refer to water by 400：1 volume ratio dilutes 84 thimerosals.
- 4. lotus rhizome stem-tip tissue Initial culture method according to claim 1, it is characterised in that step 3）Middle sterilization method For：A. with 75% alcohol disinfecting 30 seconds；B. effective chlorine density is 1% liquor natrii hypochloritis, is sterilized 30 minutes；C. it is standby after aqua sterilisa cleaning.
- 5. lotus rhizome stem-tip tissue Initial culture method according to claim 1, it is characterised in that step 3）Described in dissect The multiplication factor of mirror is 10-40x, in order to clearly observe growing point, convenient sampling.
- 6. according to the lotus rhizome stem-tip tissue Initial culture method described in claim 1, it is characterised in that step 3）It is middle sterilization and Inoculation is carried out in superclean bench.
- 7. lotus rhizome stem-tip tissue Initial culture method according to claim 1, it is characterised in that step 4）The middle training used The test tube that container is a diameter of 25mm is supported, culture medium dosage is 15-20ml.
- 8. lotus rhizome stem-tip tissue Initial culture method according to claim 1, it is characterised in that step 5）The middle training used It is 300ml triangular flasks to support container, culture medium dosage 100ml.
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| JPH0466032A (en) * | 1990-07-04 | 1992-03-02 | Mamoru Yamabe | Acclimation and mass-production of virus-freed seedling of nelumbo nucifera gaertn. |
| CN1258435A (en) * | 1999-12-22 | 2000-07-05 | 扬州大学 | Fast lotus root propagating method in detoxicated seed-breeding field |
| CN1318578C (en) * | 2004-11-26 | 2007-05-30 | 扬州大学 | Domestication method for lotus root group cultivating de-poisoning breeding |
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| CN103283595B (en) * | 2013-05-17 | 2015-04-22 | 高红胜 | Primary culture method of stem tip tissue of strawberry |
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